We propose a two-pronged approach to explore basic aspects of the entry/fusion process of herpes simplex virus (HSV) that enables virus-cell and cell-cell fusion (Aim 1) and to use that knowledge to understand how the entry glycoproteins stimulate humoral responses important in virus neutralization and protection (Aim 2). These fundamental problems apply to all herpesviruses and to other enveloped viruses. We have amassed a rich armamentarium of purified glycoprotein constructs, monoclonal antibodies (Mabs) and expression plasmids to address significant questions about the fusion pathway as well as the humoral responses in a variety of populations. The coordinated entry process requires four glycoproteins, gD, gB, a heterodimer of gH/gL, and either of two cell receptors, herpesvirus entry mediator (HVEM) and nectin-1. Each step is blocked by epitope specific virus-neutralizing Mabs. In Aim 1, we hypothesize that conformational changes to each glycoprotein convert it into a functional form. Receptor binding on one face of gD induces a conformational change to a different face that enables it to interact with gH/gL. The conformation of gH/gL is altered and its second face interacts with and activates the gB trimer to carry out fusion. We propose to solve the structures of Fab/glycoprotein complexes that define each step. We will also carry out cryo-EM studies of gB to gain insight into its prefusion form. A major biochemical approach will be to lock flexible regions involved in conformational changes using reversible disulfide bonds. We show that engineering such bonds in gD freezes conformations in place that normally have to change. Reduction of the bond restores the ability of gD to function as measured kinetically in living cells using a split luciferase assay. In Aim 2, we will use our Mabs to dissect the polyclonal responses to protein-based vaccines and in naturally HSV infected people. Some Mabs act in a synergistic fashion with others to enhance neutralization. We will discern the correlates of the humoral responses to individual epitopes of each glycoprotein that contribute to virus neutralization. To do this on larger cohorts, we have miniaturized techniques to evaluate epitope profiles and to isolate antibodies to specific proteins. These include competition analysis using the Biacore and the Octet biosensor (see letter from Dr. J. Rucker), a magnetic bead assay to capture Abs to individual glycoproteins from sera and the high-throughput Alphascreen technology that directly tests sera for their capacity to interfere with gD/receptor interactions. We have collected sera from human vaccine trials and from a large cohort of naturally infected humans. We will obtain sera from guinea pigs immunized with different combinations of the glycoproteins. Finally, our Mabs will be used in passive immunization experiments. We are collaborating with Drs. F. Rey (crystallization), A. Steven (cryo-EM), D. Bernstein and R. Cardin (guinea pig studies), A. Wald (naturally infected humans) and J. Cohen (passive immunization studies). Our proposal thus explores the basic biology of HSV entry/fusion and uses this knowledge for translational studies.